A comprehensive library of recombinant myoglobins and human hemoglobins is being generated to test key biophysical concepts concerning ligand binding and globin folding, to evaluate molecular dynamics simulations, and to provide the background data and underlying mechanisms for the rational design of more stable and efficient heme-protein based blood substitutes. Four specific aims are proposed. (1) Evaluation of the factors which regulate O2, CO, and NO affinities in Mb- The relative importance of stearic versus polar interactions will be examined by: (a) introducing mutations which alter the conformation and tautomeric structure of His(E7) and the stability of distal pocket water molecules; and (b)using IR, RR, and EPR spectroscopies to measure electrostatic potentials and stearic effects near the iron atom. (2) Determination of the structural features of kinetic barriers and intermediate states- The innermost kinetic barrier to ligand binding will be examined by measuring picosecond NO recombination in Mb mutants with varying distal pocket volume and stearic restrictions near the iron atom. The properties of the nanosecond or "C-state" intermediate will be measured indirectly by O2 geminate recombination and more directly by time resolved IR spectra of photolyzed CO. The outer kinetic barrier(s) will be explored by constructing "second shell" mutations which should block or open proposed pathways for ligand entry into the distal pocket. (3) Correlation of Apoglobin unfolding with ligand and hemin binding rates- Polar residues in the distal pocket of apomyoglobin promote unfolding of the E and B helices to form a molten globule intermediate, whereas large aliphatic or aromatic residues stabilize the native apoglobin structure. The thermodynamics of these effects will be examined by denaturant titration following intrinsic fluorescence and circular dichroism. Tryptophan mutations at key locations in the B, C, D, and F helices, and CD comer are being constructed to follow individual unfolding events. Correlations between the flexibility of these secondary structures and the speed of ligand movement through the holoprotein will be examined. (4) Regulation of ligand binding in the alpha and beta subunits of human Hb- The roles of His(E7), Val(El l), Leu(Bl0), and other key residues regulating ligand binding will be examined in recombinant Hb tetramers and isolated subunits. A key goal is to determine how R-state beta subunits promote O2 and discriminate against CO binding in the absence of hydrogen bonding with His(E7). This work requires the development of procedures for producing large amounts of functionally intact isolated subunits and of techniques for measuring T-state oxygen rate constants.